Nitric oxide and ROS mediate autophagy and regulate Alternaria alternata toxin-induced cell death in tobacco BY-2 cells

Abstract

Synergistic interaction of nitric oxide (NO) and reactive oxygen species (ROS) is essential to initiate cell death mechanisms in plants. Though autophagy is salient in either restricting or promoting hypersensitivity response (HR)-related cell death, the crosstalk between the reactive intermediates and autophagy during hypersensitivity response is paradoxical. In this investigation, the consequences of Alternaria alternata toxin (AaT) in tobacco BY-2 cells were examined. At 3 h, AaT perturbed intracellular ROS homeostasis, altered antioxidant enzyme activities, triggered mitochondrial depolarization and induced autophagy. Suppression of autophagy by 3-Methyladenine caused a decline in cell viability in AaT treated cells, which indicated the vital role of autophagy in cell survival. After 24 h, AaT facilitated Ca²⁺ influx with an accumulation of reactive oxidant intermediates and NO, to manifest necrotic cell death. Inhibition of NO accumulation by 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) decreased the level of necrotic cell death, and induced autophagy, which suggests NO accumulation represses autophagy and facilitates necrotic cell death at 24 h. Application of N-acetyl-L-cysteine at 3 h, confirmed ROS to be the key initiator of autophagy, and together with cPTIO for 24 h, revealed the combined effects of NO and ROS is required for necrotic HR cell death.

Figure 1

Alternaria alternata toxin-induced accumulation of ROS in BY-2 cells treated for 3 and 24 h. Histochemical visualization of (A) ${\mathrm{O}}_2^{\cdot -}$ generation by NBT staining and (B) graphical representation of the same. (C) Observation of ·OH, ROO· and H₂O₂ accumulation by the fluorescent probe DCFH-DA. (D) Spectroflurimetric estimation of DCF fluorescence. Scale bars denote 50 µm. Different Roman letters (3 h) or Greek letters (24 h) represent significant differences (P < 0.05) compared to control by Holm–Sidak multiple comparison test. Asterisks (*) depict the significant difference (P < 0.05) at same AaT concentration at different time points.

Figure 2

NO generation and estimation of Ca²⁺ influx induced by Alternaria alternata toxin at 3 and 24 h in BY-2 cells and consecutive effects on mitochondria and membranes. (A) Quantification of DAF-FM DA fluorescence by ImageJ software. (B) Fluorescent photomicrographs of DAF-FM DA stained BY-2 cells treated with 50 µg mL⁻¹ AaT [Scale bars denote 50 µm]. (C) Analysis of intracellular Ca²⁺ upsurge in tobacco cells. (D) Loss of mitochondrial membrane potential represented by quenching of Rhodamine 123 fluorescence [Scale bars denote 20 µm]. (E) ROS induced membrane lipid peroxidation represented by increased MDA content. Different Roman letters (3 h) or Greek letters (24 h) represent significant differences (P < 0.05) compared to control by Holm–Sidak multiple comparison test. Asterisks (*) depict the significant difference (P < 0.05) at same AaT concentration at different time points.

Figure 3

Alternaria alternata toxin-induced alterations in enzymatic and non-enzymatic antioxidant activities in tobacco BY-2 cells treated for 3 and 24 h. (A) Increase in Superoxide dismutase (SOD) activity at 24 h. (B) Increased Catalase (CAT) activity at 3 h, followed by a significant decline at 24 h. (C) Enhanced guaiacol peroxidase (GPOD) activity at 3h, followed by decreased activity after 24 h compared to that of at 3 h. (D) Dose-dependent decline in Ascorbate peroxidase (APX) activity. (E) Dose-dependent decline in Glutathione reductase (GR) activity. (F) The decrease in reduced and oxidized glutathione (GSH/GSSG) ratio. Different Roman letters (3 h) or Greek letters (24 h) represent significant differences (P < 0.05) compared to control by Holm–Sidak multiple comparison test. Asterisks (*) depict the significant difference (P < 0.05) at same AaT concentration at different time points.

Figure 4

Assessment of cell death in Alternaria alternata toxin treated tobacco BY-2 cells with positive control 10 mM H₂O₂. (A) Fluorescent photomicrographs of DAPI stained BY-2 cells treated with 50 µg mL⁻¹ AaT at 0, 3 and 24 h. (B) Flow cytometric estimation of cell cycle progression and AL-PCD like DNA fragmentation in tobacco cells, at 50 µg mL⁻¹ AaT at 0, 3 and 24 h. (C) Estimation of necrosis by lactate dehydrogenase (LDH) leakage. (D) Spectrophotometric quantification of cell death by Evans blue staining. Scale bars denote 50 µm. Graph bars with the same letters or symbols are statistically similar (P < 0.05) according to Holm–Sidak multiple comparison test. Asterisks (*) depict the significant difference (P < 0.05) at same AaT concentration at different time points.

Figure 5

Characterization of Alternaria alternata toxin-induced autophagy at 3 and 24 h using tobacco BY-2 cells expressing transgenic GFP-ATG8 protein (A–D) and by Acridine orange (AO) staining (green fluorescence [533 nm] and red fluorescence [656 nm] merged) (E–H). (A) Percentage of transgenic GFP-ATG8 tobacco BY-2 cells showing the formation of GFP-ATG8 dots as a marker of autophagy. *, ** Indicate differences between control and AaT-treated cells that are significant at the 1 and 5% levels by Holm–Sidak multiple comparison test. (B) Control cells, (C) 50 µg mL⁻¹ AaT treated cells, (D) 50 µg mL⁻¹ AaT + 10 mM 3-MA-treated GFP-ATG8 BY-2 cells. (E) Percentage of AO stained wild-type tobacco BY-2 cells showing acidic vesicles. Different Roman letters (3 h) or Greek letters (24 h) represent significant differences (P < 0.05) compared to control by Holm–Sidak multiple comparison test. Asterisks (*) depict the significant difference (P < 0.05) at same AaT concentration at different time points. (F) Control cells, (G) 50 µg mL⁻¹ AaT treated cells, (H) 50 µg mL⁻¹ AaT + 10 mM 3-Methyladenine (3-MA) treated cells stained with AO. (I) Evaluation of toxin-induced cell death and autophagy with a different combination of 100 µM NO scavenger cPTIO, 10 mM autophagic inhibitor 3-Methyladenine (3-MA) and 250 µM ROS scavenger N-Acetyl-L-cysteine (NAC). Different Roman letters, Greek letters and Roman numerals represent significant differences (P < 0.05) compared to control (3 h) by Holm–Sidak multiple comparison test.

Figure 6

Correlation among cell death, nitric oxide (NO) and autophagy in tobacco BY-2 cells after 3 h of Alternaria alternata toxin (AaT) exposure. (A) The control wild-type untreated cells, (B) 50 µg mL⁻¹ AaT, (C) 50 µg mL⁻¹ AaT + 10 mM 3-MA, (D) 50 µg mL⁻¹ AaT + 250 µM NAC treated wild-type cells stained with trypan blue, DAF-FM DA, AO and GFP-ATG8 cells. Scale bars denote 50 µm.

Figure 7

Correlation among cell death, nitric oxide (NO) and autophagy in tobacco BY-2 cells after 24 h of Alternaria alternata toxin (AaT) exposure. (A) 50 µg mL⁻¹ AaT, (B) 50 µg mL⁻¹ AaT + 100 µM cPTIO, (C) 50 µg mL⁻¹ AaT + 100 µM cPTIO + 10 mM 3-MA, (D) 50 µg mL⁻¹ AaT + 100 µM cPTIO + 250 µM NAC treated wild type cells. Scale bars denote 50 µm.